1. Bone Marrow–Derived Mesenchymal Stromal Cells from Patients with Sickle Cell Disease Display Intact Functionality 
Elizabeth O. Stenger, Raghavan Chinnadurai, Shala Yuan, Marco Garcia, Dalia Arafat, Greg Gibson, Lakshmanan Krishnamurti, Jacques Galipeau 
Biology of Blood and Marrow Transplantation 
Volume 23, Issue 5, Pages (May 2017)
DOI: /j.bbmt
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

2. Figure 1
Phenotype and function of SCD and non-SCD MSCs. (A) Low passage (P1-P3) cryopreserved SCD and non-SCD MSCs (n = 5/group) were cultured for 5 to 7 days and then analyzed using flow cytometry for the cell surface expression of markers used to identify MSCs. Data presented as histogram overlay of markers (white = SCD, light gray = non-SCD) and unstained samples (dark gray). As per International Society for Cellular Therapy guidelines [18], SCD MSCs displayed a typical MSC phenotype with >95% of cells positive for CD44, CD73, CD90, CD105, and HLA-I and <5% of cells positive for CD34, CD45, CD19, and HLA-DR. Mean fluorescence intensity (MFI) for each marker was compared between samples, with higher mean fluorescence intensity for HLA-I (P = .03), CD73 (P = .04), and CD90 (P = .01) in non-SCD samples; otherwise, no difference was seen between groups (P > .05). (B) Growth of SCD MSCs (n = 10) was compared to non-SCD MSCs (n = 5). Time in hours for MSCs to double (P0 to P1; doubling time) was calculated and was comparable between SCD (34.3 ± 10.6 hours) and non-SCD (44.1 ± 10.4 hours) samples (P = .11). (C) To evaluate the immunomodulatory function of SCD MSCs, MSCs (n = 5/group) were co-cultured for 4 days with third-party PBMCs, with or without anti-CD3/CD28 co-stimulation, and T cell proliferation was assessed by flow cytometric analysis of Ki67 expression (%CD3+Ki67+ cells). Representative flow cytometry gating strategy for a single SCD and non-SCD sample is shown. (D) When PBMCs were unstimulated, CD3+ T cells did not proliferate appreciably, including when co-cultured with MSCs (data not shown). Conversely, stimulation of PBMCs resulted in extensive proliferation of CD3+ T cells, with 63.2% having high Ki67 expression. When PBMCs were co-cultured with MSCs at varying concentrations, both SCD and non-SCD MSCs suppressed T cell proliferation in a dose-dependent manner. SCD MSCs more potently suppressed T cell proliferation at all concentrations compared with non-SCD MSCs. (E) Next, MSCs were co-cultured for 4 days with PBMCs from the same SCD donor (3 MSC and PBMC donor pairs), with or without anti-CD3/CD28 co-stimulation, and T cell proliferation was again assessed by flow cytometric analysis of Ki67 expression. Results were compared with experiments performed with non-SCD MSCs from a single donor (BMH21). All MSC samples suppressed the proliferation of SCD PBMCs in a dose-dependent manner. Statistical analysis was performed on combined data, wherein no difference was seen between SCD MSCs and the non-SCD MSC sample (P > .05 at all MSC/PBMC ratios).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

3. Figure 2
IDO as dominant pathway in suppression of T cell proliferation by SCD and non-SCD MSCs. (A) IDO gene expression was compared in unstimulated and IFN-γ–stimulated MSCs (n = 4-5/group). SCD and non-SCD MSCs were cultured overnight and then either left untreated or stimulated with IFN-γ for 4 hours. The level of IDO expression was analyzed by quantitative reverse transcriptase PCR and reported as fold induction. Unstimulated MSCs had negligible expression of IDO (non-SCD, 1.01 ± .08; SCD, 1.01 ± .07), whereas IFN-γ stimulation resulted in significant upregulation of IDO by both non-SCD (47,887 ± 8420) and SCD (22,019 ± 7879) MSCs. There was no difference in IDO expression between non-SCD and SCD MSCs. (B) To evaluate the role of contact-independent pathways (including IDO) in the immunomodulatory function of SCD MSCs, MSCs (n = 5/group) were co-cultured in a transwell system for 4 days with third-party PBMCs, with or without anti-CD3/CD28 co-stimulation, and T cell proliferation was assessed by flow cytometric analysis of Ki67 expression (%CD3+Ki67+ cells). Despite being co-cultured in a transwell system, both SCD and non-SCD MSCs continued to suppress T cell proliferation, with more potent suppression by SCD MSCs. (C) To evaluate the contribution of IDO in this contact-independent suppression of T cell proliferation, co-culture experiments were repeated ± 1-methyl-DL-tryptophan (1MT). Representative flow cytometry gating strategy for a single SCD and non-SCD sample is shown. (D) In the presence of 1MT, both SCD and non-SCD MSCs lost their ability (compared to without 1MT) to suppress T cell proliferation, with proliferation comparable to stimulated T cells without the addition of MSCs.
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions

4. Figure 3
Molecular signature of SCD and non-SCD MSCs in response to IFN-γ. Expression of 47 hematopoiesis genes was assessed using a Fluidigm array in unstimulated and IFN-γ–stimulated MSCs from individuals with and without SCD (n = 4-5/group). (A) Cycle threshold (CT) for each gene in each MSC sample is depicted as a heat map, where red denotes high expression and blue denotes low expression. Each sample was duplicated, as indicated by _1 or _2 after the sample ID, where SCD indicates a SCD sample and BMH indicates healthy control MSC. Gene names are indicated across the top of the heat map. Cluster analysis was performed by Ward's method and demonstrated no clustering by MSC donor source (clustering predominantly by IFN-γ stimulation status). (B) Volcano plots contrast the significance (negative logarithm of the P value, high values more significant) against the difference in average cycle threshold value. This plot shows that only 4 hematopoiesis genes were significantly differentially expressed in SCD versus non-SCD MSCs (P < .001, allowing Bonferroni adjustment for 47 comparisons), although the magnitude of the difference was small (range, −2.8 to +1.0). (C) On the contrary, 24 hematopoiesis genes were found to be significantly differentially expressed in unstimulated versus IFN-γ–stimulated MSCs (P < .001), most notably IDO, and with a wider magnitude of difference (range, −4.7 to +18.7).
Biology of Blood and Marrow Transplantation  , DOI: ( /j.bbmt )
Copyright © 2017 The American Society for Blood and Marrow Transplantation Terms and Conditions